Schupbach et al. (Journal of Medical Virology, 2001, 65:225-232) discloses that heat-denatured, amplification-boosted p24 antigen can be used as an alternative to HIV RNA testing in order to monitor the treatment of HIV infection. Respess et al. (Journal of Clinical Microbiology, 2005, 43(1):506-508) and Knuchel et al. (Journal of Clinical Virology, 2006, 36:64-67) also disclose ultrasensitive p24 antigen assays as an alternative to HIV RNA testing.
Boder et al. (PNAS, 2000, 97(20):10701-10705) discloses directed evolution of antibody fragments with monovalent femtomolar antigen-binding affinity. Holliger and Hudson (Nature Biotechnology, 2005, 23(9):1126-1136) reviews engineered antibody fragments. Nygren and Uhlen (Current Opinion in Structural Biology, 1997, 7:463-469) and Hosse et al. (Protein Science, 2006, 15:14-27) review engineering of protein display scaffolds for molecular recognition.
Binz et al. (Nature Biotechnology, 2005, 23(10):1257-1268) and Hey et al. (Trends in Biotechnology, 2005, 23(10):514-422) review engineering of novel binding proteins from nonimmunoglobulin domains.
Bi-specific recombinant antibody molecules that can recognize and bring together two different ligands are well-known in the literature (see e.g., Albrecht et al., J Immunol Meth 310: 100-16, 2006). Bi-specific recombinant antibodies that bind to two different epitopes in the same protein have also been described (see e.g. Neri et al., J Mol Biol 246: 367-73, 1995; Zhou, J Mol Biol 329: 1-8, 2003). The combinatorial binding resulted in a significant increase in binding affinity compared to binding of each of the two recombinant antibodies alone. While MEBIPs may have similarities with construction of bi-specific recombinant antibodies, it is important to appreciate that the present innovation is novel and unrelated to the described design and use of bi-specific recombinant antibodies.
Although helpful, the increased affinity involved in cooperative binding of more than one covalently joined BHAP (whether a recombinant antibodies or another type of molecule) is not the reason for targeting multiple regions in the protein of interest. Instead, the key idea of this innovation is to combine scattered short conserved peptides within a variable protein to a “virtual epitope” that provides sufficient complexity for diagnostic specificity in detection. To provide such structural complexity a linear peptide epitope should consist of at least six residues. However, a look into available sequence databases shows that well conserved continuous 6-residues amino acids stretches are hard to find in many highly variable microbial proteins, in particular those of RNA viruses. For example, the HIV-1 p24 protein does not contain a single hexapeptide (6-mer) that would be conserved in more than 99% of known HIV-1 sequences, which makes its reliable immunological detection problematic. As discussed below, coordinated detection of combinations of conserved tri-, tetra-, or pentapeptides using the MEBIP approach can help to solve this problem. Thus, this novel approach therefore allows development of better means for diagnostic detection of highly variable microbial proteins, such as HIV-1 p24.